1,3-Propanediol is a monomer useful in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds.
A variety of chemical routes to 1,3-propanediol are known. For example, 1,3-propanediol is prepared from 1) ethylene oxide over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid; 2) by the catalytic solution phase hydration of acrolein followed by reduction; or 3) from hydrocarbons such as glycerol, reacted in the presence of carbon monoxide and hydrogen over catalysts having atoms from Group VIII of the periodic table. Although it is possible to generate 1,3-propanediol by these chemical methods, they are expensive and generate waste streams containing environmental pollutants.
It has been known for over a century that 1,3-propanediol can be produced from the fermentation of glycerol. Bacterial strains able to produce 1,3-propane-diol have been found, for example, in the groups Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. In each case studied, glycerol is converted to 1,3-propanediol in a two-step, enzyme-catalyzed reaction sequence. In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HP) and water (Equation 1). In the second step, 3-HP is reduced to 1,3-propanediol by a NAD+-linked oxidoreductase (Equation 2).Glycerol→3-HP+H2O  (Equation 1)3-HP+NADH+H+→1,3-Propanediol+NAD+  (Equation 2)The 1,3-propanediol is not metabolized further and, as a result, accumulates in high concentration in the media. The overall reaction consumes a reducing equivalent in the form of a cofactor, reduced β-nicotinamide adenine dinucleotide (NADH), which is oxidized to nicotinamide adenine dinucleotide (NAD+).
The production of 1,3-propanediol from glycerol is generally performed under anaerobic conditions using glycerol as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors. For example, in strains of Citrobacter, Clostridium, and Klebsiella, a parallel pathway for glycerol operates under these conditions which first involves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD+- (or NADP+-) linked glycerol dehydrogenase (Equation 3). The DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase (Equation 4), becomes available for biosynthesis and for supporting ATP generation via, for example, glycolysis.Glycerol+NAD+→DHA+NADH+H+  (Equation 3)DHA+ATP→DHAP+ADP  (Equation 4)In contrast to the 1,3-propanediol pathway, this pathway may provide carbon and energy to the cell and produces rather than consumes NADH.
In Klebsiella pneumoniae and Citrobacter freundii, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon. The dha regulons from Citrobacter and Klebsiella have been expressed in Escherichia coli and have been shown to convert glycerol to 1,3-propanediol.
The biological production of 1,3-propanediol requires glycerol as a substrate for a two-step sequential reaction in which a dehydratase enzyme (typically a coenzyme B12-dependent dehydratase) converts glycerol to an intermediate, 3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by a NADH- (or NADPH) dependent oxidoreductase. The complexity of the cofactor requirements necessitates that a whole cell catalyst be used for an industrial process incorporating this reaction sequence for the production of 1,3-propanediol. A process for the production of 1,3-propanediol from glycerol using an organism containing a coenzyme B12-dependent diol dehydratase is described in U.S. Pat. No. 5,633,362 (Nagarajan et al.). However, the process is not limited to the use of glycerol as feedstock. Glucose and other carbohydrates are suitable substrates and, recently, these substrates have been shown to be substrates for 1,3-propanediol production. Carbohydrates are converted to 1,3-propanediol using mixed microbial cultures where the carbohydrate is first fermented to glycerol by one microbial species and then converted to 1,3-propanediol by a second microbial species U.S. Pat. No. 5,599,689 (Haynie et al.). However, a single organism able to convert carbohydrates to 1,3-propanediol is preferred for reasons of simplicity and economy. Such an organism is described in U.S. Pat. No. 5,686,276 (Laffend et al.); and in U.S. Ser. No. 60/030,601 and U.S. Ser. No. 08/969,683.
Glycerol dehydratase and diol dehydratase are coenzyme B12-dependent enzymes which catalyze the conversion of glycerol to 3-HP (Toraya, T., In Metalloenzymes Involving Amino Acid-Residue and Related Radicals; Sigel, H. and Sigel, A., Eds.; Metal Ions in Biological Systems; Marcel Dekker: New York, 1994; Vol. 30, pp 217-254). Coenzyme B12 may be provided by the whole cell catalyst through de novo synthesis. However, if the coenzyme B12 requirement of the B12-dependent dehydratases exceeds the de novo synthesis capacity of the whole cell catalyst or if the whole cell catalyst lacks the de novo synthesis capacity, then coenzyme B12 or coenzyme B12 precursors may be provided in the reaction medium. Due to the cost and instability of coenzyme B12, medium supplementation with coenzyme B12 precursors is preferred; and this then requires the conversion of these precursors to coenzyme B12. In addition, glycerol dehydratase and diol dehydratase undergo inactivation which involves loss of the 5′-deoxyadenosyl moiety from coenzyme B12 and the formation of hydroxocobalamin and/or cob(II)alamin. (Toraya, T., supra.) Thus, readenosylation of hydroxocobalamin and/or cob(II)alamin is required for the recycling of coenzyme B12.
Vitamin B12 (cyanocobalamin) and hydroxocobalamin are stable, commercially available coenzyme B12 precursors which are readily taken up by microorganisms. Conversion of these precursors, both Co(III) species, to coenzyme B12 (5′-deoxyadenosyl cobalamin) involves: 1.) reduction of Co(III) to Co(II) (i.e., formation of cob(II)alamin by a aquacobalamin reductase), 2.) reduction of Co(II) to Co(I) (i.e., formation of cob(I)alamin by a cob(II)alamin reductase), and 3.) ATP-dependent adenosylation of cob(I)alamin by a cob(I)alamin adenosyltransferase to form coenzyme B12 Enzymes associated with these functions have been described for Salmonella typhimurium, Pseudomonas denitrificans, and Clostridium tetanomorphum. Suh and Escalante-Semerena, J. Bacteriol. 177, 921-925 (1995) and references therein. Similar systems have been described for Euglena gracilis (Watanabe et al., Arch. Biochem. Biophys. 305, 421-427 (1993)), Chlamydomonas reinhardtii (Watanabe et al., Biochim. Biophys. Acta 1075, 36-41 (1991)), and mammalian cells (Pezacka, E. H., Biochim. Biophys. Acta 1157, 167-177 (1993)).
The problem to be solved is how to biologically produce 1,3-propanediol by a single recombinant organism containing genes facilitating the synthesis of B12 coenzyme in the presence of a B12-dependent dehydratase enzyme.